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Assessing the agricultural reuse of the digestate from microalgae anaerobic 2

digestion and co-digestion with sewage sludge 3

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Maria Solé-Bundó1, Mirko Cucina 1,2 Montserrat Folch3, Josefina Tàpias3, Giovanni Gigliotti 2, 5

Marianna Garfí 1, Ivet Ferrer1,* 6

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1 GEMMA - Environmental Engineering and Microbiology Research Group, Department of Civil 9

and Environmental Engineering, Universitat Politècnica de Catalunya·BarcelonaTech, c/ Jordi 10

Girona 1-3, Building D1, E-08034, Barcelona, Spain 11

2 Department of Civil and Environmental Engineering, University of Perugia, Borgo XX Giugno 74, 12

06124 Perugia, Italy 13

3 Department of Natural Products, Plant Biology and Soil Science, School of Pharmacy, University 14

of Barcelona, Avda. Joan XXIII s/n, E-08028 Barcelona, Spain 15

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* Corresponding author: 18

Tel.: +34 93 401 64 63 19

Fax: +34 934017357 20

E-mail address: ivet.ferrer@upc.edu 21

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Solé-Bundó, M., Cucina, M., Folch, M., Tapias, J., Gigliotti, G., Garfí, M., Ferrer, I.* (2017) Assessing the 23

agricultural reuse of the digestate from microalgae anaerobic digestion and co-digestion with 24

sewage sludge. Science of the Total Environment 586 (1-9) 25

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Abstract 26

Microalgae anaerobic digestion produces biogas along with a digestate that may be reused in 27

agriculture. However, the properties of this digestate for agricultural reuse have yet to be 28

determined. The aim of this study was to characterise digestates from different microalgae 29

anaerobic digestion processes (i.e. digestion of untreated microalgae, thermally pretreated 30

microalgae and thermally pretreated microalgae in co-digestion with primary sludge). The main 31

parameters evaluated were organic matter, macronutrients and heavy metals content, hygenisation, 32

potential phytotoxicity and organic matter stabilisation. According to the results, all microalgae 33

digestates presented suitable organic matter and macronutrients, especially organic and ammonium 34

nitrogen, for agricultural soils amendment. However, the thermally pretreated microalgae digestate 35

was the least stabilised digestate in comparison with untreated microalgae and co-digestion 36

digestates. In vivo bioassays demonstrated that the digestates did not show residual phytotoxicity 37

when properly diluted, being the co-digestion digestate the one which presented less phytotoxicity. 38

Heavy metals contents resulted far below the threshold established by the European legislation on 39

sludge spreading. Moreover, low presence of E.coli was observed in all digestates. Therefore, 40

agricultural reuse of thermally pretreated microalgae and primary sludge co-digestate through 41

irrigation emerges a suitable strategy to recycle nutrients from wastewater. 42

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Keywords: 44

Anaerobic co-digestion; Biofertiliser; Biogas; Land spreading; Phytotoxicity; Thermal pretreatment 45

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1. Introduction 46

Microalgae-based wastewater treatment systems represent a cost-effective alternative to 47

conventional activated sludge systems. The major advantage is that mechanical aeration is not 48

required, since oxygen is provided by microalgae photosynthesis. Moreover, microalgae cultures 49

are capable of removing nutrients (N, P) from wastewater by means of different mechanisms, such 50

as assimilation or precipitation (Rawat et al., 2011). Furthermore, these systems can also combine 51

wastewater treatment and bioenergy production if harvested microalgal biomass is downstream 52

processed. In particular, anaerobic digestion is one of the most well-known processes to valorise 53

organic waste generated in a wastewater treatment plant. Over the last decades, several studies on 54

biogas production from microalgae have been carried out (Uggetti et al., 2017). They have 55

demonstrated that some microalgae species have a resistant cell wall, which may hamper their 56

bioconversion into methane. Microalgae cell wall disruption could be enhanced by applying 57

pretreatment methods, being the most suitable those pretreatments with low energy demands 58

(Passos et al., 2014). Besides, in the context of microalgae grown in wastewater, co-digestion of 59

microalgae with sewage sludge is a profitable strategy, since the sludge is generated in the same 60

process chain (Uggetti et al., 2017). This could optimise waste management and increase the 61

organic loading rate of the digester (Mata-Alvarez et al., 2014). 62

Apart from biogas, microalgae anaerobic digestion also produces a digestate that can be 63

reused in agriculture. Even though several studies have pointed out the necessity of recycling 64

nutrients through digestate reuse to improve the sustainability of biogas production from microalgae 65

(Collet et al., 2011), the properties of microalgae digestate for agricultural reuse have yet to be 66

characterised. In general, anaerobic digestates have proper chemical properties for agricultural reuse 67

(Rowell et al., 2001). For instance, they are rich in ammonia nitrogen, readily available for plant 68

uptake, and other macronutrients such us phosphorus and potassium (Teglia et al., 2011a). However, 69

depending on digestates properties, their reuse could be more addressed to improve or maintain the 70

physico-chemical or biological properties of soils (soil amendment) or to boost the plants growing 71

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(fertilisers). In the first case, digestates with high organic matter, organic carbon and organic 72

nitrogen content are preferred, while digestates with important mineral fractions have a higher 73

potential for application as fertiliser (Nkoa, 2014). 74

Anaerobic digestion is often designed to achieve the maximum energy production, leading 75

to a low stabilisation of the organic matter of the feedstock. As a consequence, digestates may be 76

characterised by a high labile organic matter content and, thus, their agricultural reuse may face 77

agronomic and environmental issues. In fact, it is known that by adding low-stabilised organic 78

matter the soil microbial activity may be excessively stimulated. Indeed, it can produce high CO2 79

fluxes from the soil, soil oxygen consumption with sequential nitrogen losses, and phytotoxicity 80

phenomena (Pezzolla et al., 2013; Abdullahi et al., 2008). In addition, the digestate composition can 81

highly vary depending on the feedstock or anaerobic digestion operating conditions. Even the 82

application of a pretreatment on the feedstock previous to anaerobic digestion can influence the 83

final composition of the digestate (Monlau et al., 2015a). Thus, the characterisation of a digestate 84

before evaluating its potential applications is convenient. 85

When characterising new digestates, particular attention should be addressed to the 86

macronutrients content, potential phytotoxicity and stabilization of the organic matter. In vivo 87

bioassays are useful to assess the potential phytotoxicity (Alburquerque et al., 2012; Zucconi et al., 88

1985). The quantification of CO2 emissions and the water extractable organic matter (WEOM) in 89

digestate amended soils are suitable strategies to assess organic matter stabilization (Pezzolla et al., 90

2013; Said-Pullicino and Gigliotti, 2007). On the other hand, land application of anaerobic 91

digestates may also introduce physical, chemical and biological contaminants into soils which may 92

be up-taken by crops and endanger their long-term agricultural activity (Nkoa, 2014). For instance, 93

European legislation on sewage sludge spreading (EC Directive 86/278/CEC) mainly regulates the 94

heavy metals content in digestates to avoid their accumulation in amended soils. However, a more 95

recent European Directive draft (2003/CEC) also proposes restrictions on the occurrence of bio-96

accumulative organic compounds and their hygenisation before being spread on soils. 97

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Consequently, the presence of these contaminants in digestates should be assessed if they are going 98

to be reused in agricultural soils. 99

The aim of this study was to characterise for the first time the quality of microalgae 100

digestates for agricultural reuse. To this end, the effluents from three different anaerobic digesters 101

fed by untreated microalgae, thermally pretreated microalgae and thermally pretreated microalgae 102

in co-digestion with primary sludge were analysed. The main parameters evaluated were organic 103

matter, macronutrients and heavy metals content, hygenisation, potential phytotoxicity and organic 104

matter stabilisation. 105

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2. Material and Methods 107

2.1 Digestate origin and sampling 108

The microalgal biomass used in this study consisted of a microalgae-bacteria consortia grown in a 109

pilot raceway pond that treated wastewater from a municipal sewer, as described by (Passos et al., 110

2015). Microalgal biomass was harvested from secondary settlers and gravity thickened in 111

laboratory Imhoff cones at 4 ºC for 24 hours. The pilot plant was located at the laboratory of the 112

GEMMA research group (Barcelona, Spain). According to optic microscope examinations (Motic 113

BA310E, equipped with a camera NiKon DS-Fi2), predominant microalgae were Chlorella sp. and 114

diatoms (Fig. 1). 115

In order to improve microalgae biodegradability, a part of the harvested and thickened 116

biomass was thermally pretreated at 75 ºC for 10h, as suggested by Passos and Ferrer (2014). The 117

pretreatment of microalgal biomass was carried out in glass bottles with a total volume of 250 mL 118

and a liquid volume of 150 mL, which were placed in an incubator under continuous stirring at 75 119

ºC for 10h. Untreated (control) and pretreated microalgae were digested in lab-scale reactors under 120

mesophilic conditions. Furthermore, the anaerobic co-digestion of pretreated microalgal biomass 121

with primary sludge (25%-75% VS, respectively) was also evaluated. The thickened primary sludge 122

was collected in a municipal wastewater treatment plant near Barcelona. 123

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Thus, the following effluents from microalgae anaerobic digestion were analysed: 124

• Digester 1 (D1): Microalgal biomass; 125

• Digester 2 (D2): Thermally pretreated microalgal biomass; 126

• Digester 3 (D3): Co-digestion of pretreated microalgal biomass and primary sludge. 127

Anaerobic reactors (1.5 L) were operated on a daily feeding basis, where same volume was purged 128

from and added to digesters using plastic syringes. Operation conditions of the reactors and 129

feedstock characteristics are shown in Table 1. Digestate samples were analysed weekly over a 130

period of 11 weeks of stable reactors operation. Physico-chemical properties were analysed during 131

11 weeks (n=11) while macronutrients and pathogens were analysed during the last 6 weeks (n=6) 132

and the heavy metals during the 3 last weeks (n=3). 133

134

2.2 Digestate characterisation 135

2.2.1. Physicochemical properties and macronutrients 136

Total solids (TS), volatile solids (VS), total chemical oxygen demand (COD) and total Kjeldahl 137

nitrogen (TKN) were analysed according to Standard Methods (APHA, 2005). Ammonium nitrogen 138

(NH4+-N) was measured according to the Solorzano method (Solorzano, 1969). Volatile fatty acids 139

(VFA) concentrations were measured by injecting 1 µL of centrifuged (4200 rpm for 8 min) and 140

filtered samples (0.2 µm) into an Agilent 7820A GC after sulphuric acid and diisoprppyl ether 141

addition. The GC was equipped with an auto-sampler, flame ionization detector and a capillary 142

column (DP-FFAB Agilent 30 m x 0.25 mm x 0.25 µm), and operated at injector and detector 143

temperatures of 200 and 300ºC, respectively, with helium as carrier gas. Electric conductivity (EC) 144

was determined with a Crison EC-Meter GLP 31+ and pH with a Crison Portable 506 pH-meter. 145

Total organic carbon (TOC) and total nitrogen (TN) were measured using an automatic analyser (aj- 146

Analyzer multi N/C 2100S). TOC was analysed with an infrared detector (NDIR) according to 147

combustion-infrared method of Standard Methods (APHA, 2005) by means of catalytic oxidation at 148

800 ºC using CeO2 as catalyst. Following, a solid-state chemical detector (ChD) was used to 149

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quantify TN as NOx. Phosphorous was determined by means of Olsen-P modified method 150

(Watanabe and Olsen, 1965). Ca+2 and Mg+2 were analysed by EDTA titrimetric method after 151

ammonium acetate extraction (1N at pH 7), while Na+ and K+ were determined by flame 152

photometric method after ammonium acetate extraction (1N at pH 7) (MAPA, 1994). 153

Dewaterability was evaluated by means of the capillary suction time (CST) test (Triton Electronics 154

Ltd.). 155

2.2.2. Heavy metals 156

In order to determine the heavy metals concentration, samples were dried at 100ºC during 24h. 157

After HCL-HNO3 (3:1, v/v) digestion (200ºC, 15 min) of dry digestate, Cd, Cr, Cu, Hg, Ni, Pb and 158

Zn were determined by inductively coupled plasma mass spectrometry (ICP-MS) (Perkin Elmer 159

Elan 6000). 160

2.2.3. Pathogens 161

Escherichia coli (E. coli) was determined according to Standard Methods (APHA, 2005). The E. 162

coli ChromIDTM Coli (COLI ID-F) used in this study was supplied by Biomérieux and the culture 163

medium was m-coliBlue24® from Difco. 164

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2.3 Organic matter stabilisation 166

2.3.1 Soil incubation procedure 167

Organic matter stabilisation from digestates was evaluated through a microcosm soil experiment. 168

Fresh digestates were used to amend an agricultural soil (soil chemical characterization not shown), 169

using a digestate dose according to the limits prescribed by the European Nitrates Directive 170

(91/676/CEC) for the protection of groundwater against pollution caused by nitrates. Specifically, 171

digestate application doses were calculated to apply 170 kg N ha-1. 200g of soil (dry matter) were 172

amended and placed in an incubation chamber (20 ±2°C) for 30 days at 70% of the water holding 173

capacity. 174

2.3.2 CO2 emissions evaluation 175

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CO2 emissions resulting from the organic matter mineralization were measured after 0, 2, 5, 8, 12, 176

20 and 30 days of amending, using an alkaline-trap and subsequent titration. At the same time, 10g 177

(fresh weight) of soil were collected and air-dried for the WEOM determination. 178

2.3.3 Water extractable organic matter determination 179

The WEOM was analysed both in the digestates and amended soils. Fresh digestate samples were 180

centrifuged at 4,200 rpm for 6 min and filtered through a 0.45 µm membrane filter (GVS). Soil 181

WEOM was extracted from the dry soil samples with deionised water (solid to water ratio of 1:10 182

w/w) for 24 h. The suspensions were then centrifuged at 4,200 rpm for 6 min and filtered through a 183

0.45 µm membrane filter. Water Extractable Organic Carbon (WEOC) concentration in the filtrates 184

was then measured by an automatic analyser (Analytic Jena-Analyzer multi N/C 2100S) and the 185

WEOM was calculated according the following equation (Pribyl, 2010): 186

WEOM = WEOC · 2.0 187

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2.4 Potential phytotoxicity 189

2.4.1. Seed germination bioassay 190

To evaluate the germination index (GI), a modified phytotoxicity test employing seed germination 191

was used (Zucconi et al., 1985). Pure digestates together with three dilutions (0.1%, 1% and 10% 192

v/v in deionised water) were used as germination media. A filter paper placed inside a 9 cm 193

diameter Petri dish was wetted with 1 mL of each germination solution and 10 Lepidium sativum L. 194

seeds were placed on the paper. 100% deionised water was used as a control. Five replicates were 195

set out for each treatment. The Petri dishes, closed with plastic film to avoid moisture loss, were 196

kept in the dark for 2 days at 20 °C. After the incubation period, the number of germinated seeds 197

and the primary root length were measured. The GI was expressed as a percentage of the control. 198

2.4.2. Plant growth bioassay 199

To evaluate the influence of digestate on plant biomass accumulation, a modified phytotoxicity test 200

employing plant growth was used (Alburquerque et al., 2012). Plastic seedbeds made of 12 cells (50 201

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mL/cell with a drainage hole in the bottom) were used for the experiment, after filling them with 202

commercial perlite (2-3 mm diameter). Seedbeds were placed 24 h in a vessel (20x15x5 cm) 203

containing 500 mL of deionised water to reach the saturation of the substrate. Then, 5 seeds of 204

Lepidium sativum L. were sown in each cell. After the 3 days needed for the germination and 205

seedlings occurrence, 32 seedlings were left in each seedbed and deionised water was replaced by 206

500 mL of the digestate dilutions to be tested (0.1 %, 1% and 10% v/v). Pure digestates were not 207

tested in this case, since no germination was observed in the germination test. One seedbed was 208

used as a control, leaving 100% deionised water as growth media. During all the experiment, the 209

vessels were placed in environmental controlled conditions (25±2°C, daily photoperiod of 14 h). At 210

the end of the experiment, after 10 days from the replacement of the growth media, seedlings 211

survived were harvested and their total dry mass (TS) was determined after drying at 105°C. The 212

growth index (GrI) was calculated for each digestates as the percentage of the control (distilled 213

water). The whole experiment was replicated three times. 214

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3. Results and Discussion 216

3.1 Physico-chemical characterisation 217

All the digestates analysed presented low dry matter content (~3% TS) (Table 2) and can be 218

considered as liquid products. To ease their management, these digestates could be directly spread 219

on soils in nearby areas. However, if transportation/distribution was required, a dewatering process 220

to reduce the moisture content would be recommended. If we look at the CST measurements, which 221

estimate the ability of each digestate to release water (Gray, 2015), we can see how microalgae 222

digestates presented poor dewaterability (25 and 28 s·gTS-1·L for D1 and D2, respectively), while 223

these results were consistently improved by the co-digestion of primary sludge (8 s·gTS-1·L) (Table 224

2). This is due to the higher dewaterability of primary sludge digestate with respect to microalgae 225

digestate. 226

On the other hand, the measured pH presented slightly-alkaline values in all digestates 227

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(>7.0). Among them, pretreated microalgae digestate (D2) presented the highest pH value, which 228

can be attributed to the higher concentration of NH4+-N released from proteins during the thermal 229

pretreatment (Passos and Ferrer, 2014). However, all pH values are compatible with the common 230

pH on soils and therefore, their application should not affect the soil pH. 231

Other factors that may cause an impact on soils after digestate spreading are the EC and 232

VFA’s content, since phytotoxicity effects have been correlated to both parameters (Alburquerque et 233

al., 2012; Di Maria et al., 2014). Although EC was moderate in all digestates (5.9-8.2 dS·m-1), the 234

digestate from the co-digestion showed the lowest value. Consequently, it would cause less impact 235

on soil. Besides, all digestates showed low VFA’s concentrations (Table 2). Again, the lowest value 236

was found in the co-digestion digestate (10 mgCOD-eq·L-1). This indicates that the anaerobic 237

digestion process results in a more stabilised digestate when pretreated microalgae are co-digested 238

with the primary sludge. 239

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3.2 Organic matter and fertiliser properties 241

The three digestates had moderate organic content due to organic matter mineralization during the 242

anaerobic digestion process. While the two microalgae digestates presented a similar VS/TS ratio of 243

53-54%, the percentage of organic matter in the co-digestion digestate was lower (47%) due to the 244

higher mineralization of primary sludge, which is a more readily biodegradable substrate than 245

microalgae. In fact, the percentage of organic matter in digestates is highly dependent on the type of 246

substrate and the operating conditions of anaerobic reactors (Monlau et al., 2015b). For instance, 247

Teglia et al. (2011a) compared digestates from different origins and found that digestates from agri-248

food industries showed higher organic matter content than digestates from sewage treatment plants. 249

The results obtained in this study are in accordance with those from similar microalgae anaerobic 250

digestion processes (Passos and Ferrer, 2014, 2015). 251

Several studies have shown that anaerobic digestates can be as effective as mineral fertilisers 252

(Nkoa, 2014). To assess the fertiliser properties of the microalgae digestates, the macronutrients 253

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content was here evaluated (Table 3). The main nutrient present in all digestates was nitrogen. Even 254

so, the nitrogen content of microalgae digestates (both untreated and thermally pretreated) was 255

significantly higher than the co-digestion digestate (39-42%), showing values of 80 g·kg TS-1 and 256

56 g·kg TS-1, respectively. Microalgae digestates presented similar nitrogen values compared to 257

those from farm-byproducts that are frequently applied as nitrogen suppliers on soils (Alburquerque 258

et al., 2012; Zucconi et al., 1985). Moreover, the nitrogen content was much higher than the 259

common values found in sewage sludge digestates (36-40 g·kg TS-1) (Di Maria et al., 2014; Gell et 260

al., 2011), even in the co-digestion digestate. The highest concentration of NH4+-N was found in the 261

pretreated microalgae digestate. However, the NH4+-N/TKN ratio only varied from 30.9 to 33.8% 262

among all digestates, presenting all of them a similar soluble mineral nitrogen fraction. This means 263

that the organic nitrogen fraction is predominating in all digestates, so they should be used as soil 264

amendment rather than fertiliser (Teglia et al., 2011b). As expected, the digestates also showed low 265

C/N ratios around 3 (Table 2). These values are within the typical range for other digestates as 266

sewage sludge, poultry slurry or pig slurry (Alburquerque et al., 2012; Gutser et al., 2005). 267

Unfortunately, with low C/N ratios, N is present in excess and it can be lost by ammonia 268

volatilization or leaching (Bernal et al., 2009). In order to increase the carbon content in microalgae 269

digestates, they could be co-digested with other carbon rich substrates, like waste paper (Yen and 270

Brune, 2007). 271

Moderate quantities of P and K+ were also found in all the digestates (Table 3). P content 272

was slightly higher in microalgae digestates (D2 and D3) compared to the digestate obtained by the 273

co-digestion (3.6-3.9 and 3.2 g P·kg TS-1, respectively). On the other hand, the content of K+ of the 274

microalgae digestates was 2-fold higher compared to the digestate obtained by the co-digestion 275

(4.8-5.2 and 2.2 g K·kg TS-1, respectively). Conversely to nitrogen, no significant differences were 276

found between P and K+ contents of microalgae and sewage sludge digestates. In particular, 277

literature reported values from 2.2-3.0 g K·kg TS-1 and 3.2-3.8 g P·kg TS-1 in sewage sludge 278

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digestates (Di Maria et al., 2014; Gell et al., 2011; Tambone et al., 2010), which fall within the 279

range of the co-digestion digestate analysed in the present study. Ca2+, Mg2+ and Na+ presented 280

similar concentrations in all the cases. This can be attributed to the composition of the wastewater 281

treated in both systems where microalgae and primary sludge were obtained, which came from the 282

same water source. The content of salts should be carefully analysed when applying the digestates 283

to the soils to avoid their salinization, especially the presence of Na+ (Daliakopoulos et al., 2016). 284

On the whole, microalgae digestates could especially contribute to nitrogen supply on soils. 285

However, with a moderate NH4+-N/TKN ratio (<35%) their use should be addressed as soil 286

amendment rather than direct biofertiliser. Indeed, the digestates nutrients content was lower than 287

those recommended by the standards of European countries that have regulated the commercial uses 288

of liquid fertilisers (EC 2003/2003). Conversely, their organic matter content and their high mineral 289

and organic nitrogen content make them suitable for land spreading. Nonetheless, the stability of 290

organic matter and potential toxicity of digestates must be taken into account, along with their 291

potential risks on soil contamination. These issues are analyzed and discussed in the following 292

sections. 293

294

3.3 Stabilisation of the organic matter 295

Figure 2a shows the CO2 emissions measured from the digestate amended soils studied in 296

the microcosm experiment. Whereas the control (un-amended soil) showed moderately constant 297

emission rates throughout the incubation period, the addition of digestates increased the CO2 fluxes 298

with respect to the control, particularly in the first days after amendment. Similar results were 299

obtained by other authors after amending soils with anaerobic digestate and compost (Alluvione et 300

al., 2010; Pezzolla et al., 2013). The highest emission rates were observed immediately after 301

applying the digestates for the soils treated with pretreated microalgae (D2) and co-digestion (D3) 302

digestates (230 and 245 mgCO2 kgdm-1

d-1, respectively). CO2 emissions decreased steadily over 303

time, reaching constant values similar to the control ones within 13 days. Conversely, the soil 304

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treated with unpretreated microalgae (D1) showed a different behaviour, whose highest value was 305

observed after 2 days from the amendment (170 mgCO2 kgdm-1

d-1). Besides, cumulative net CO2 306

emissions at the end of the incubation period increased in the following order: D1 < D3 < D2 (Table 307

4). Considering the amount of organic carbon added to the soil with the microalgae digestates 308

(Table 4), higher fluxes of CO2 were expected from D1 and D3 amended soils. However, the 309

highest cumulative CO2 emissions were detected for the soil amended with thermally pretreated 310

microalgae, indicating that the organic matter of this digestate was less stabilised than the organic 311

matter of the other digestates (D1 and D3). This is in accordance with the fact that D1 and D3 also 312

showed lower biodegradability in the soil than D2. It can be deducted from the values of C-313

mineralization, expressed as the % of the added TOC that was mineralised at the end of the 314

incubation (Table 4). The lower stabilisation of pretreated microalgae digestate with respect to the 315

other digestates could be attributed to the different anaerobic digesters operations. For instance, 316

comparing the anaerobic digestion of unpretreated and thermally pretreated microalgal biomass, 317

higher NH4+-N and VFA concentrations were found in the latter (Passos and Ferrer, 2014). As a 318

consequence, the digestate from thermally pretreated microalgae could be less stabilised and could 319

show higher soluble organic matter content that can be quickly mineralized in the soil. On the other 320

hand, the co-digestion with primary sludge could also reduce the NH4+-N and VFA concentrations 321

in the reactors. The addition of easily degradable substances to the soil implies the consumption of 322

soil oxygen that, in some circumstances, can lead to anoxic conditions, fermentation processes and 323

to the production of phytotoxic substances (Wu et al., 2000). Stability-dependent respiration rates 324

were reported by various authors for soils amended with organic materials (Sánchez-Monedero et 325

al., 2004). Most of them also observed CO2 emissions peaks in the first few days after amendment 326

with an intensity related to the contents of WEOM and microbial biomass. In fact, it is well known 327

that organic amendment can change the amount and quality of dissolved organic matter present in 328

the soil solution (Chantigny, 2003). As WEOM is an easily available organic matter fraction for soil 329

microorganisms, it has important implications on microbial activity and soil respiration. Moreover, 330

14

Said-Pullicino et al., (2007) have shown that the soluble organic matter fraction of organic 331

amendments tends to decrease with organic matter stabilisation. 332

Figure 2b shows the time course of the WEOM in the digestate amended soils. Digestate 333

application enhanced significantly (P < 0.05) the concentration of WEOM in the treated soils with 334

respect to control during the first days after amendment. Following, the WEOM concentration 335

showed a clear decreasing trend during the incubation period due to the soil microbial respiration. 336

While D1 and D3 amended soils showed a decrease of WEOM content to the control level, in the 337

D2 amended soils the WEOM mineralisation appears to be stronger and lead to a final content 338

significantly lower (P < 0.05) than the control soils. The WEOM behaviour observed in the D2 339

amended soils and the low biodegradability showed by D1 and D3 appear to be in contrast with the 340

WEOM concentrations in the microalgae-derived digestates (Table 4). In fact, D1 showed a higher 341

content of WEOM with respect to D2 and D3. Therefore, it can be assumed that the labile organic 342

matter of D2 was characterized by a low stability due to the thermal pretreatment of the microalgae 343

biomass that was responsible for the solubilisation of labile and reactive organic compounds. As a 344

consequence, the application of the thermal pretreated microalgae digestate to the soil can lead to 345

the priming effect, with strong short-term changes in the turn-over of soil organic matter after the 346

application of low stabilized organic amendments (Kuzyakov et al., 2000). 347

In all the amended soils, the strongest WEOM mineralization appeared to be concluded after 348

13 days from the application, similarly to what was observed for the CO2 emissions. As already 349

demonstrated by Pezzolla et al. (2013), when an organic amendment is applied to soil, WEOM is 350

strictly related to the soil CO2 emission rates. In the present work, this fact was confirmed by the 351

correlation between the soil respiration rates of all the soil samples and their WEOM contents. 352

Indeed, a high positive correlation was found (y = 1.5313x - 2655.5) to be significant (r = 0.7750) at 353

P < 0.05 (n = 28). In the last two weeks of incubation a constant trend was observed for the WEOM 354

content in the amended soils. This behaviour can be explained considering the dynamic equilibrium 355

15

that occurs between the consumption of WEOM due to the mineralization and the release of 356

WEOM by the soil microorganism during their hydrolytic activity (Rochette and Gregorich, 1998). 357

In the light of the results obtained, it appears clear that pretreated microalgae digestate is 358

less recommendable for soil application than the other digestates due to the low stabilisation of its 359

soluble organic matter. Indeed, untreated microalgae and co-digestion digestates spreading lead to a 360

lower impact on soil system and higher benefits for the environment and the agriculture. 361

362

3.4 Evaluation of the potential phytotoxicity of digestates 363

Phytotoxicity effects are often found in anaerobic digestates due to the high contents in soluble 364

salts, NH4+-N and low weight organic compounds (i.e. volatile fatty acids, phenols) (Alburquerque 365

et al., 2012). In this study, the GI was used to evaluate the digestates phytotoxicity by applying 366

different concentrations of digestate (100%, 10%, 1% and 0.1%) and comparing the germination of 367

cress seeds (Lepidium sativum L.) to a control (100% of deionised water) (Fig. 3). 368

The results showed that no germination was detected for any pure digestate. Thus, the GI of 369

pure digestates (0%) indicates that they cannot be spread on agricultural soils without dilution or a 370

stabilisation post-treatment process. For instance, a composting post-treatment would produce a 371

compost where phytotoxic compounds, still abundant in anaerobic digestates and responsible of the 372

absence of germination (Abdullahi et al., 2008), can be reduced. Conversely, positive results in the 373

germination assays were found for digestate dilutions. Untreated and pretreated microalgae 374

digestates (D1 and D2, respectively) gave a similar GI trend, showing the highest GI for the 0.1% 375

dilution (109.9% and 97.3%, respectively). At this dilution (0.1%), the highest GI was observed for 376

D1, probably due to the lower content of ammonia nitrogen with respect to D2 (Table 3). In both 377

cases, the lowest GI value was observed at 10% dilution. On the contrary, no significant differences 378

were observed between 1% and 0.1% dilutions, when values close to the control were achieved. It 379

means that the largest phytotoxic potential was removed at 1% dilution. Concerning D3, there were 380

no significant (P < 0.05) differences for the GI between dilutions of 10%, 1% and 0.1% (GI of 381

16

97.8%, 109.5% and 101.9% respectively), meaning that the phytotoxicity effect of the microalgae 382

digestate was reduced through the co-digestion. Indeed, co-digestion processes are known to be 383

more advantageous than mono-digestion ones due to a dilution effect of inhibitory compounds, 384

among other factors (Tritt, 1992). 385

Moreover, the effect of digestates dilutions (10%, 1% and 0.1%) on the biomass production 386

of cress (Lepidium sativum L.), expressed as GrI, were evaluated (Fig. 4). Concerning D1, no 387

significant (P < 0.05) phytotoxic effect was detected on the production of biomass. Conversely, D2 388

showed a strong reduction of GrI at the highest concentration tested (10%), which is probably due 389

to the high content of ammonium nitrogen of D2 (Table 3). At lower concentrations (1%, 0.1%), the 390

GrI of D2 increased due to the dilution of the phytotoxic compounds. For both D1 and D2, the 1% 391

dilution which showed a significantly higher (P < 0.05) GrI than the 0.1% dilution. As shown for 392

other plants, low level of phytotoxicity can lead to a normal growth, or even higher than the un-393

stressed control, due to the genetic adaptability of the plants (Wang et al., 2015). This phenomena 394

may be responsible of the GrI behaviours in D1 and D2. Nevertheless, the best performance in the 395

plant growth bioassay was obtained from D3. Thus, co-digestion process appears to be the most 396

suitable process for the reduction of phytotoxicity as already showed by the results obtained from 397

the GI bioassay. Concerning the GrI determination, 10% and 1% dilutions of D3 did not show 398

significant differences with respect to the control, showing the absence of residual phytotoxicity. 399

When diluted at 0.1%, D3 showed plant nutrient, growth stimulant or even phytohormone-like 400

effects (Alburquerque et al., 2012) that lead to a significant increase of the GrI (P < 0.05) with 401

respect to the control (128.1%). 402

In the present work, NH4+-N, VFA and EC of the digestates were found to be significantly 403

(P < 0.05) and negatively correlated both to GI and GrI, as expected from what described in 404

literature (Alburquerque et al., 2012; Zucconi et al., 1985). Statistical models used in this evaluation 405

are described in Table 5. 406

In light of what was found in the germination and growth bioassays, agricultural application 407

17

of the microalgae-derived digestates through dilution in the irrigation water would be the most 408

suitable option, as the digestate would be diluted before coming in contact with seeds and plants. 409

Moreover, dilution could also avoid salts and heavy metal concentration in the soil (Moral et al., 410

2005). Co-digestion digestate appeared to be the most suitable for agricultural reuse. In fact, it 411

would require less water for dilution and, thus, it would be a more concentrated organic fertiliser. 412

Moreover, the co-digestion digestate was the only one that did not show residual phytotoxicity; 413

conversely it showed stimulating properties in the in vivo assays. 414

415

3.5 Potential risks of digestates: heavy metals and pathogens 416

In order to assess the potential risks of soil contamination after digestate spreading, the occurrence 417

of heavy metals and the presence of pathogens (E. Coli) were evaluated. 418

Concerning heavy metals, their concentrations in the three digestates were lower than the 419

threshold established by the sludge European Directive (EC directive 86/278/CEC), and also by the 420

even more restrictive EU Directive draft (2003/CEC) (Table 6). Although all digestates presented 421

appropriate heavy metal contents for soil application, special attention should be paid to the co-422

digestion digestate because of its high Zn content that is originated from the primary sludge. This is 423

a particularity of the wastewater treatment plant where the primary sludge was collected, since they 424

receive wastewater from industries generating high Zn concentration in their effluents. With regards 425

to the microalgae digestate, despite microalgae ability for assimilating metals (Suresh Kumar et al., 426

2015), no significant heavy metal concentrations increase was found in microalgae digestates (D1 427

and D2) compared to the mixture with the primary sludge (D3) (Table 6). 428

Regarding the digestate hygenisation, low E.coli presence was found in all digestates (Table 429

7), below the threshold values proposed by the EU Directive draft on spreading sludge on land (less 430

than 5·105 colony forming units per gram of wet weight of treated sludge) (2003/CEC). Moreover, 431

it is noteworthy that thermal pretreatment improved the hygenisation leading to absence of E.coli in 432

the digestate. In fact, according to the EU draft, the combination of thermal pretreatment and 433

18

anaerobic digestion can be considered as an advanced sludge treatment. 434

435

4. Conclusions 436

Agricultural reuse of the digestate from microalgae anaerobic digestion and co-digestion with 437

primary sludge appears to be a promising solution towards zero waste generation in microalgae-438

based wastewater treatment systems. All microalgae digestates considered in this study presented 439

organic matter and macronutrients content, especially organic and ammonium nitrogen, suitable for 440

agricultural soils amendment. However, the thermal pretreated digestate presented a higher 441

concentration of easily consumable organic carbon that can be mineralized on soil producing 442

environmental impacts. Conversely, untreated microalgae and co-digestion digestates appeared to 443

be more stabilised. In vivo bioassays demonstrated that the digestates did not show residual 444

phytotoxicity when properly diluted, being the co-digestion digestate the one which presented less 445

phytotoxicity. Furthermore, it showed interesting stimulant properties for plants. Heavy metals 446

contents resulted far below the threshold established by the European legislation on sludge 447

spreading. Low presence of E.coli was observed in all digestates. In addition, the thermal 448

pretreatment improved the hygenisation obtaining absence of E.coli in the digestate. In this context, 449

agricultural reuse of thermally pretreated microalgae and primary sludge co-digestate through 450

irrigation emerges as a suitable strategy to recycle the nutrients and organic matter in agriculture. 451

452

Acknowledgements 453

This research was funded by the Spanish Ministry of Science and Innovation (project 454

FOTOBIOGAS CTQ2014-57293-C3-3-R). Maria Solé is grateful to the Universitat Politècnica de 455

Catalunya·BarcelonaTech for her PhD scholarship. Marianna Garfí is grateful to the Spanish 456

Ministry of Economy and Competitiveness (Plan Nacional de I+D+i 2008-2011, Subprograma Juan 457

de la Cierva (JDC) 2012). The authors would like to acknowledge Humbert Salvadó from the 458

University of Barcelona for the valuable help on microalgae microscopic images and 459

19

characterisation. 460

461

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24

Table 1. Main parameters of the anaerobic digestion and feedstock properties. 590

Digester 1 (D1):

Microalgae

Digester 2 (D2):

Pretreated microalgae

Digester 3 (D3):

Co-digestion

Operation conditions

Temperature (ºC) 36.2 ± 1.1 36.6 ± 1.8 35.7 ± 1.8

OLR (gVS/L.day) 0.83 ± 0.04 0.82 ± 0.02 0.83 ± 0.01

HRT (days) 30 30 30

Feedstock

Composition (% VS) 100 % MB 100 % P-MB 25 % P-MB + 75% PS

TS (%) 3.9 ± 0.4 3.7 ± 0.3 3.7 ± 0.4

VS (%) 2.5 ± 0.2 2.4 ± 0.1 2.4 ± 0.1

VS/TS (%) 66 ± 5 66 ± 6 66 ± 8

COD (g/L) 43.4 ± 8.1 44.0 ± 7.0 48.1 ± 8.0

Note: OLR= organic loading rate, HRT= hydraulic retention time, TS= total solids, VS= volatile solids, COD= chemical 591 oxygen demand, MB= microalgal biomass; P-MB= pretreated microalgal biomass, PS= primary sludge. 592

Pretreatment conditions: 75ºC, 10h. 593

25

Table 2. Main physico-chemical properties and organic matter of the three microalgae digestates 594

analysed (mean ± SD; n=11, except for TOC and TN (n=3)). 595

Parameter Units Digestate D1:

Microalgae

Digestate D2:

Pretreated microalgae

Digestate D3:

Co-digestion

pH - 7.35a ± 0.11 7.55b ± 0.08 7.30a ± 0.15

EC dS·m-1 7.0b ± 0.7 8.2a ± 0.3 5.9c ± 0.4

TS g·g-1,% 3.0a ± 0.1 2.9a ± 0.2 3.0a ± 0.2

VS g·g-1,% 1.6b ± 0.1 1.5b ± 0.1 1.4a ± 0.1

VS/TS % 54b ± 2 53b ± 1 47a ± 2

COD g·L-1 26a ± 2 25a ± 2 24a ± 1

TOC g·L-1 7.6 ± 0.1 6.4 ± 0.0 6.1 ± 0.1

TN g·L-1 2.4 ± 0.0 2.2 ± 0.1 1.9 ±0.1

C/N - 3.17 2.98 3.27

VFA mgCOD-eq·L-1 100a ± 138 270a ± 365 10a ± 25

CST s 795b ± 71 919b ± 122 272a ± 21

s·gTS-1·L 25b ± 3 28b ± 4 8a ± 1

Note: TS= total solids, VS= volatile solids, COD= chemical oxygen demand, TOC= total organic carbon, TN= total 596 nitrogen, C/N= Carbon-Nitrogen ratio, VFA= volatile fatty acids, CST= capillary suction time 597

a,b,c letters indicate a significant difference between digestates at a level of p < 0.05 after Tuckey’s test. 598

26

Table 3. Macronutrients characterisation of the three digestates analysed (mean ± SD, n=6). 599

Parameter Units Digestate D1:

Microalgae

Digestate D2:

Pretreated microalgae

Digestate D3:

Co-digestion

TKN gN·L-1 2.4a ± 0.1 2.3a ± 0.1 1.7b ± 0.0

gN·kg TS-1 79.8a ± 4.0 80.6a ± 2.2 56.0b ± 1.1

NH4+-N gN·L-1 0.7b ± 0.1 0.8b ± 0.1 0.5a ± 0.1

NH4+-N/TKN % 30.9 33.8 32.5

P

gP2O5·L-1 0.25b ± 0.02 0.27b ± 0.02 0.21a ± 0.03

gP·kg TS-1 3.6b ± 0.3 3.9b ± 0.2 3.2a ± 0.5

K

gK2O·L-1 0.17b ± 0.03 0.19b ± 0.02 0.08a ± 0.03

gK·kg TS-1 4.8b ± 0.8 5.2b ± 0.7 2.2a ± 1.0

Ca

gCaO·L-1 0.43a ± 0.13 0.37a ± 0.10 0.54b ± 0.07

gCa·kg TS-1 10.2a ± 3.1 8.9a ± 2.4 13.4b ± 1.7

Mg gMgO·L-1 0.18a ± 0.09 0.21a ± 0.09 0.17a ± 0.10

gMg·kg TS-1 3.6a ± 1.8 4.2a ± 1.8 3.6a ± 2.0

Na

gNa2O·L-1 0.40b ± 0.05 0.38b ± 0.06 0.32a ± 0.03

gNa·kg TS-1 10.0b ± 1.3 9.4b ± 1.4 8.1a ± 0.8

Note: TKN= total Kjeldahl nitrogen 600 a,b letters indicate a significant difference between digestates at the level of p < 0.05 after Tuckey’s test. 601

27

Table 4. Carbon mineralization rate from digestate amended soils after 30 days of incubation (mean 602

± SD, n=3). 603

Parameter Units Digestate D1:

Microalgae

Digestate D2:

Pretreated microalgae

Digestate D3:

Co-digestion

Total N* mg·L-1 2.4± 0.1 2.2± 0.1 1.9± 0.2

Application dose mL 13.0 14.3 16.6

TOCadded mg 98.1 92.0 101.1

WEOM mg·L-1 1335.9 892.3 790.5

WEOM added mg 17.4 12.8 13.1

Net CO2 emission mg-C 21.2± 1.9 47.1± 2.1 30.7± 2.6

TOCadded mineralized % 21.6 ± 1.7 51.2 ± 6.7 30.4 ± 5.2

Note: TOC= total organic carbon, WEOM= water extractable organic matter 604 *: total N values used for the dosage calculation 605

606

28

Table 5. Linear regression equations (y = mx + q) calculated for selected parameters of the 607 digestates (n=11). 608

Y x m q r

N-NH4+ GI -0.0073 0.7254 0.9054*

VFA -0.6728 67.351 0.9301*

EC -0.0067 6.7041 0.9572*

N-NH4+ GrI -0.0068 0.6826 0.8691*

VFA -0.6270 63.0660 0.8862*

EC -0.0628 6.2935 0.9156*

Note: GI= Germination Index, GrI= Growth Index, VFA= volatile fatty acids, EC= electric conductivity 609 *: significant at P < 0.05 610

29

Table 6. Concentration of heavy metals in microalgae digestates (mean+SD, n=3). 611

Parameter Units Digestate D1:

Microalgae

Digestate D2:

Pretreated microalgae

Digestate D3:

Co-digestion

Limit values*

Limit values**

Cd mg·kg TS-1 2.2 a ± 1.9 2.7 a ± 0.3 8.6 a ± 5.4 20-40 10

Cu mg·kg TS-1 584 a ± 108 593 a ± 100 491 a ± 23 1000-1750 1000

Pb mg·kg TS-1 47 a ± 3 49 a ± 1 221 b ± 112 750-1200 750

Zn mg·kg TS-1 637 a ± 53 592 a ± 9 2202 b ± 135 2500-4000 2500

Ni mg·kg TS-1 104 a ± 9 127 a ± 9 101 a ± 5 300-400 300

Cr mg·kg TS-1 69 a ± 2 75 a ± 14 127 b ± 9 - 1000

Hg mg·kg TS-1 2.0 a ± 0.5 1.7 a ± 0.6 <1.1 a ± 0.2 16-25 10

*: Limit values according to current European legislation (EC directive 86/278/CEC) 612 **: Limit values according to the European draft (2003/CEC) 613

a,b letters indicate a significant difference between digestates at the level of p < 0.05 after Tuckey’s test. 614

30

Table 7. Escherichia coli content (CFU/ml) in microalgae digestates (mean ± sd; n=6). 615

Digestate Mean Maximum value

D1 (microalgae) 39.8 316.2

D2 (pretreated microalgae) 0.0 Absence

D3 (co-digestion) 25.1 199.5 Note: CFU= colony forming units 616

31

617

Figure 1. Microscopic image of microalgal biomass mainly composed by Chlorella sp. and diatoms. 618

619

32

a) 620 621

b) 622 623

624

Figure 2. (a) CO2 emissions from microalgae-derived digestates amended soil (mean+SD, n=3); (b) 625

Water extractable organic matter content in microalgae-derived digestates amended soil during the 626

incubation period (mean±SD, n=3). Results are expressed on soil dry matter basis. 627

628

50

100

150

200

250

300

0 2 5 8 13 20 30

mgC

O2

kgdw

-1d-

1

Time (days)

Control

D1 (microalgae)

D2 (pretreated microalgae)

D3 (co-digestion)

3500

3700

3900

4100

4300

4500

4700

4900

5100

5300

5500

0 5 10 15 20 25 30

mg

WE

OM

kgdw

-1

Time (days)

Control

D1 (microalgae)

D2 (pretreated microalgae)

D3 (co-digestion)

33

629 630

Figure 3. Effects of microalgae digestates and their dilutions on the germination index (GI) of cress 631

(Lepidium sativum L.) (mean+SD, n=5). GI was 0% for all the pure (100%) digestates. 632

0

20

40

60

80

100

120

140

10% 1% 0.1%

GI (

% o

f the

con

trol

)

D1 (microalgae)

D2 (pretreated microalgae)

D3 (co-digestion)

34

633 634 Figure 4. Effects of microalgae digestates and their dilutions on the growth index (GrI) of cress 635

(Lepidium sativum L.) (mean+SD, n=5). 636

0

20

40

60

80

100

120

140

10% 1% 0.1%

GrI

(%

of t

he c

ontr

ol)

D1 (microalgae)D2 (pretreated microalgae)D3 (co-digestion)